1. Field of the Invention
The present invention relates to microbiological industry, and specifically relates to a method for producing amino acids. More specifically, the present invention concerns the use of a new feedback-resistant enzyme involved in cysteine biosynthesis. More specifically, the present invention concerns a new feedback-resistant mutant serine acetyltransferase, E. coli strains harboring the enzyme, and a method for producing L-cysteine by fermentation using the strains.
2. Description of the Related Art
Conventionally, L-amino acids have been industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources or mutants of the said strains which have been specially modified to enhance L-amino acid productivity.
Many techniques to enhance L-amino acid productivity, for example, by transformation of microorganism with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765) have been disclosed. These techniques are based on increasing the activity of the enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes from feedback inhibition by produced L-amino acid (see, for example, Japanese Laid-open application No 56-18596 (1981), WO 95/16042 or U.S. Pat. Nos. 5,661,012 and 6,040,160).
The biosynthesis of L-cysteine from L-serine in E. coli bacteria is performed by serine acetyltransferase, encoded by the cysE gene, and O-acetylserine (thiol)-lyase isozymes designated-A and -B, encoded by cysK and cysM genes. Serine acetyltransferase (also called “SAT”; EC 2.3.1.30) catalyzes the formation of O-acetyl-L-serine from acetyl-CoA and L-serine and plays a regulatory role in the biosynthesis of cysteine via feedback inhibition by L-cysteine (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996).
A mutant of SAT from Escherichia coli without feedback sensitivity to L-cysteine, and wild-type SAT were isolated by Denk D. and Bock A. (J. Gen. Microbiol., 1987, 133 (Pt 3), 515-25). The mutant cysE gene showed a single base change in position 767 resulting in a substitution of methionine at position 256 to isoleucine. This mutant excreted L-cysteine.
Either replacement of wild-type Met-256 with 19 other amino acid residues in E. coli SAT encoded by cysE gene or introduction of a termination codon to truncate 256-273 C-terminal region leads, in most cases, to feedback resistant (fbr) phenotype The mutant SAT proteins, however, do not restore the level of activity of wild-type SAT (Nakamori S. et al, Appl. Environ. Microbiol., 1998, 64, 5, 1607-1611, WO 97/15673). Strains harboring a plasmid having these altered cysE genes produced up to 200 mg/l of cysteine, including cystine.
Numerous feedback-insensitive mutant SATs from E. coli were obtained by PCR via random mutagenesis. Mutations were identified along the entire amino acid sequence of SAT, but all the mutant SATs demonstrated a significantly decreased specific activity level (Takagi, H. et al, FEBS Lett., 1999, 452, 323-327).
The essential role of the C-terminal region of the SAT in the feedback inhibition by L-cysteine was also shown by Mino K. et al (Biosci. Biotechnol. Biochem. 1999, 63, 1, 168-179). The truncated SAT, which is the wild type SAT cleaved between Ser 253 and Met 254, resulting in a deletion of 20 amino acid residues from the C-terminus, was much less sensitive to feedback inhibition than the wild-type SAT.
It has also been reported that L-cysteine can be produced by a bacterial strain belonging to the genus Escherichia, characterized by both a suppressed L-cysteine decomposition system, e.g. a lower activity of cysteine desulfhydrase, and the retention of SAT having decreased feedback inhibition by L-cysteine, e.g. a mutant SAT with the 256th methionine at position 256 in the wild-type SAT substituted with another amino acid residue (JP11155571A2).
A recombinant serine acetyltransferase having a mutation in the sequence region of amino acids 97-100, 164-169, 237, 239-240, 245-259 and 267-269 or a deletion in the C-terminal sequence region of amino acids 237-240, 245-259 and 267-269 exhibits reduced sensitivity to L-cysteine in comparison to the wild-type enzyme, and is disclosed in US patent 6,218,168. The following mutations in SAT, encoded by double mutant cysEXIV allele, showed good resistance to cysteine (Ki>1000 μM) with relatively high activity (0.453 μM/min×mg): A to G at position 721, resulting in a change of Thr167 to Ala167 and ATG to TAG at positions 988-990, resulting in a stop-codon instead of Met256. Strain JM15, transformed with the cysEXIV allele, produced 2.3 g/l of L-cysteine after 48 hours of feed-batch fermentation process. The best yield of L-cysteine (3.9 g/l), however, was obtained using cysEDe1—255 mutant allele, whereby the 18 amino acids at the C-terminal region were truncated (U.S. Pat. No. 6,218,168).
As a rule, the fbr phenotype of an enzyme arises as a result of replacing one or more amino acid residues with other amino acid residues in protein sequence, and these replacements lead to reducing the enzyme activity.
The disadvantage of the mutant enzymes obtained by the methods described above, therefore, is the decreased activity of the mutant enzymes in comparison with the wild-type enzymes. Clearly there is a need in the art for maintainance of the mutant enzyme activity in the fbr phenotype.